Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically foc...
Graphical abstract Design of experiments (DOE) is an appropriate approach for probe sequence optimization in real-time PCR.Nine MP designs led to maximum information for the 3 input factors.Using DOE the maximum number of individual reactions performed was 180.A one-factor-at-a-time approach in...
OligoArchitect(sigma.com/probedesignonline)提供了自身二聚体和交叉二聚体杂交强度的详细信息(图9.1)。由引物与其自身或与其配偶体杂交形成的任何3'-末端二聚体必须非常弱(ΔG≥ -2.0 kcal,图9.1)。 如何减少引物二聚体形成? 应避免任何具有末端ΔG < -2.0 kcal、同时又具有可延伸的3'-末端(5'-重叠)的...
Most of the reviews on PCR optimization (Erlich et al., 1991; Dieffenbach 1993; Roux 1995) consider different parameters of PCR but generally do not discuss basic concepts of PCR primer design. Because of the requirements for different strategies of PCR, more effective PCR studies would be atta...
NewQuantiTect™ Probe PCR and RT-PCR Kits — minimize your PCR optimization for highly sensitive results!This article describes a highly sensitive competitive RT-PCR assay to accurately quantify mRNA levels of a tumor marker. Use of OmniscriptTM RT and the QIAGEN(R) Taq PCR Master Mix Kit ...
Use this as a general guideline, but note that optimization may still be necessary. GC content: As with primer sequences, aim for a GC content of 35–65% and avoid a G at the 5’ end to prevent quenching of the 5’ fluorophore. Primer and probe design considerations Complementarity ...
Validating Primer Design Optimizing Probe Concentration Optimizing Reaction Components and Multiplex Assays Optimizing Mg2+ Concentration Probe Fluorophore and Quencher Selection Guidelines for Optimization of Quantitative Reverse Transcription PCR (RT-qPCR) Assay Evaluation Optimizing PCR Conditions Assay optimizatio...
cDNA synthesis step using reverse transcriptase, followed by a PCR step using a thermostable DNA polymerase. This Kit combines Reverse Transcriptase (MMLV-RTase) and RNase Inhibitor in a single mixture, with hotstart Taq DNA polymerase in a separate 2x reaction mix optimized for probe based qRT-...
The advantages of using dsDNA-binding dyes include simple PCR primer design (only two sequence-specific DNA primers are needed, so probe design is not necessary), the ability to test multiple genes quickly without the need for multiple probes (for example, in the validation of gene expression...
Accordingly, for future studies there is potential for optimization in primer and probe design, reagent concentrations and/or reaction conditions (Svec et al., 2015). Both assays achieved a low inter- and inter-assay variability (EHV-2: <1.41% in all dilutions; EHV-5: <2.96% in all ...